Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells

Abstract

Recent studies have demonstrated that PH domains specific for PI(3,4,5)P3 accumulate at the leading edge of a number of migrating cells and that PI3Ks and PTEN associate with the membrane at the front and back, respectively, of chemotaxing Dictyostelium discoideum cells. However, the dependence of chemoattractant induced changes in PI(3,4,5)P3 on PI3K and PTEN activities have not been defined. We find that bulk PI(3,4,5)P3 levels increase transiently upon chemoattractant stimulation, and the changes are greater and more prolonged in pten- cells. PI3K activation increases within 5 s of chemoattractant addition and then declines to a low level of activity identically in wild-type and pten- cells. Reconstitution of the PI3K activation profile can be achieved by mixing membranes from stimulated pi3k1-/pi3k2- cells with cytosolic PI3Ks from unstimulated cells. These studies show that significant control of chemotaxis occurs upstream of the PI3Ks and that regulation of the PI3Ks and PTEN cooperate to shape the temporal and spatial localization of PI(3,4,5)P3.

Figure 1.

Characterization of PH_Crac binding to the PIs. (A) Purified PH_Crac-GFP-His proteins were used to probe the PVDF membrane onto which eight specific phosphoinositides were spotted. They are 1) PI(3,4,5)P₃, 2) PI(3,4)P₂, 3) PI3P, 4) PI(4,5)P₂, 5) PI4P, 6) PI, 7) PC, and 8) PS. Each spot represents 0.2 μg of lipid. (B) The purified PH_Crac-GFP-His proteins were used to probe the PVDF membrane onto which seven different concentrations of PI(3,4,5)P₃ and PI(3,4)P₂ were spotted. They are 200, 40, 20, 10, 5, 2.5, and 2 pm (1–7, respectively). (C) The ability of various inositol phosphates and PI(3,4,5)P₃ to compete with [³H]Ins(1,3,4,5)P₄ for binding to PH_Crac was compared using a PEG precipitation method. The concentrations of various inositol phosphates and PI(3,4,5)P₃ used were plotted against percentage of [³H]Ins(1,3,4,5)P₄ bound. Data represent average of three independent experiments

Figure 2.

Changes in the level of PI(3,4,5)P₃ in response to chemoattractant. (A) PI3K phosphorylates the 3′ position of PI(4,5)P₂ to produce PI(3,4,5)P₃. The in vivo and in vitro assays measure the activation of PI3K using ATPγ³²P as substrate and PI(3,4,5)P₃-³²P as a readout or using PH_Crac-GFP binding to reflect the relative amount of PI(3,4,5)P₃ and PI(3,4)P₂ produced. The assay that measures the relative amounts of PI(3,4,5)P₃ involves labeling the intact cells with ³²P. (B) The fold increase in the relative amounts of PI(3,4,5)P₃ in both wild-type and pten^– cells in response to cAMP. Wild-type and pten^– cells were metabolically labeled with ³²P for 1 h. The cells were stimulated with 10 μM cAMP, and the lipids were extracted and analyzed by TLC. Image J was used to quantify the PI(3,4,5)P₃ band intensities. The intensity values of PI(3,4,5)P₃ band were normalized to wild-type cells at time 0. The experiment was repeated three times.

Figure 6.

Reconstitution of PIP₃ production with wild-type supernatant. (A) Wild-type and pi3k1^–/pi3k2^– cells were stimulated with 10 μM cAMP. At specific time points, aliquots of cells were lysed and incubated with pi3k1^–/pi3k2^– supernatant containing PH_Crac-eGFP. The incubation allowed the PI(3,4,5)P₃ production, which was measured by PH_Crac-eGFP bound to the membrane. The membrane fraction was isolated and blotted for PH_Crac-eGFP using GFP antibody. The data were normalized to that at time 0. (B) Amount of PH_Crac-eGFP that becomes associated with membrane fractions followed chemoattractant stimulation as described in (A) except the supernatant was wild-type supernatant containing PH_Crac-eGFP. (C) The cell lysates of wild-type and pi3k1^–/pi3k2^– were stimulated with GTPγS. The cell lysates were incubated with supernatant as indicated in the panel. Image J was used to quantify the band intensities of PH_Crac-eGFP. The GTPγS treated samples were normalized to those of w/o GTPγS treatment. All the graphs in this figure are the average of two independent experiments.

Figure 3.

PI3K activation elicited by chemoattractant stimulation. (A) PI3K activation of wild-type, pten^– and pi3k1^–/pi3k2^– cells elicited by cAMP. The cells were lysed at specific time points after cAMP stimulation. The cell lysates were incubated with ATPγ³²P for 30 s, and lipids were extracted and analyzed by TLC. The bands corresponding to PI(3,4,5)P₃ were cut from plate and counted in scintillation vials. The Y-axis is cpm generated in 30 s, which represents the rate of activation. The graph represents average of three independent experiments. (B) PI3K activation of wild-type, Myr-PIK1, and Myr-PIk2 cells after cAMP stimulation. The cells were processed as described in Figure 3A. The Y-axis represents values of the band intensities of PI(3,4,5)P₃ quantified using Image J. The graph represents average of two independent experiments

Figure 4.

Activation of PI3K in response to GTPγS in various cell lines and the effect of LY294002 on PI3K activation. (A) PI3K activation of wild-type, pten^– and pi3k1^–/pi3k2^– cells. The cell lysates of these three different cell lines were treated with GTPγS, and the cell lysates were incubated with ATPγ³²P for 3 min. The lipids were extracted and were analyzed by TLC plate. Image J was used to measure the intensities of the PI(3,4,5)P₃ bands. The data of activated level (w GTPγS) were normalized to the basal level (w/o GTPγS). The graph represents the average of three independent experiments. (B) The effect of different concentrations of LY294002 on PI3K activation in wild-type cells. Wild-type cell lysates were either incubated with no LY294002 (C) or with 10, 30, 50, and 100 μM of LY294002, respectively (1–4), in the presence or absence of GTPγS. The graph represents the average of two independent experiments

Figure 5.

PI3K activation in different mutant cell lines and adaptation of PI3K. (A) PI3K activation in g_α2^–, g_β^–, and yakA^– null cells. Cell lysates of wild-type, g_α2^–, g_β^–, and yakA^– cells were stimulated with GTPγS and processed as described in Figure 4A. The experiment was repeated twice. (B) Adapted wild-type cells show a reduced PI3K activation. Wild-type cells were either treated with buffer (1) or with 10 μM cAMP every 2 min for 20 min (2). The untreated and persistently treated cells were lysed and stimulated with GTPγS. TLC was processed with Image J as described in Figure 4A. The graph represents three independent experiments.

Figure 7.

The schematic diagram demonstrating the new mechanisms of PI3K activation. At resting state, the sites (gray rectangle) on the membrane are incapable of recruiting and activating PI3Ks (blue symbol) in the cytosol. After stimulation of cells with cAMP or cell lysates with GTPγS, the binding/activation sites (pink rectangle) appear on the membrane, which recruit and activate PI3Ks (blue symbol). However with persistent treatment of chemoattractant, these sites decline. The further activation of adapted cell by GTPγS cannot generate these binding/activation sites.